Sulfated hyaluronic acid and esters thereof

ABSTRACT

Hyaluronic acid, hyaluronate esters and salts thereof are sulfated such that the number of sulfate groups per monomeric unit is in the range of from 0.5 to 3.5. The sulfated derivatives exhibit anticoagulant and cell adhesion reduction properties, and may be used to prepare biomaterials.

BACKGROUND OF THE INVENTION

This application is a divisional of copending application Ser. No.09/126,135, filed on Jul. 30, 1998 now U.S. Pat. No. 6,051,701, which isa Divisional Application of application Ser. No. 08/553,290 filed onFeb. 8, 1996, now U.S. Pat. No. 6,027,241 the entire contents of whichare hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the homogeneous sulfation ofpolysaccharides and semisynthetic derivatives thereof, in particularglycosaminoglycans such as hyaluronic acid and its esters andtetraalkylammonium salts, for the preparation of new biomaterials usefulin biomedical, health care, and pharmaceutical applications, and to suchbiomateriale per se. Such sulfated derivatives exhibit anti-thromboticactivity as evidenced by the lengthening of both the thrombin time andthe whole blood clotting time. Moreover, the absence of hemolysis andthe growth and shape of endothelial cells placed in contact with suchsulfated derivatives indicate that these materials are promisingheparin-like compounds.

DESCRIPTION OF THE RELATED ART

Many molecules of biological origin are polyelectrolytes, and theirinteractions are very important in a wide variety of biochemicalreactions. Consequently, synthetic and/or semisynthetic polyelectrolyteshave been in use for some time now. These polyelectrolytes mimic thebiological characteristics of natural polyelectrolytes, and can havesomewhat different characteristics compared to the starting material.

Polyelectrolytes of biological origin include sulfated polysaccharides,and in particular, heparin and its derivatives (D. A. Lane and U.Lindahl, Eds., Heparin-Chemical and Biological Properties, ClinicalApplications, Edward Arnold, London), which play an important role incell-substrate interactions, particularly in the process of viralactivity inhibition, in the process of blood coagulation, in lipidremoval, etc.

Heparin is the most biologically reactive member of the family ofsulfated glycosaminoglycans. It is well known for its antithrombotic andanticoagulant properties. In fact, it is extensively used in themanagement of cardiovascular diseases anti contributes enormously to thesuccess of open heart surgery. Nevertheless, the structure of heparin isnot simple and, due to the number of variations, is not entirely known.Commercial heparins consist of a spectrum of 21 heparins (Nader et al.(1974) Biochem. Biophys. Res. Commun. 57:488) ranging in molecularweights from 3,000 to 37,500 in varying anticoagulant activities.

The blood anticoagulant activity of heparin is attributed to structuralfeatures, e.g., degree of sulfation, degree of dissociation, particularsequences of COO⁻ and SO⁻ ₃ groups, as well as to molecular shape andsize. These factors appear to be related to biological activity byvirtue of their importance in the ion binding capacity of heparin(Stivala et al. (1967) Arch. Biochem. Biophys. 122:40). By virtue of itshighly negatively charged nature, heparin has a strong affinity forcations, and its activity is pH-dependent.

Most of the readily available natural polysaccharides have been sulfatedin an attempt to obtain heparin analogues (Hoffman et al. (982)Carbohydrate Res. 2:115; Kindness et al. (1980) Brit. J. Pharmac.63:675; Horton et al. (1973) Carbohydrate Res. 30:349; Okada et al.(1979) Makromol. Chem. 180:813; Kikuchi et al. (1979) Nippon KagakuKaishi 1:127; Manzac et al. (1981) Proc. Third M.I.S.A.O. 5:504), andrecently, sulfate, carboxylic, and sulfonate groups were attached tosynthetic polymers such as polystyrene (Kanmaugue et al. (1985)Biomaterials 6:297) and polyurethane (Ito et al. (1992) Biomaterials13:131). The anticoagulant activities of these materials were much lowerthan that heparin, and were dependent on the type and binding of thesubstituents, the degree of substitution, and sequences.

Some chemical reactions are known which make it possible to sulfatepolysaccharides (WO 88/00211; EP 0 340 628; Nagasawa et al. (1986)Carbohydrate Research 158:183-190), but it has not yet been possible toobtain sulfated polysaccharides which, besides the chemical andchemical-physical characteristics peculiar to such polysaccharides, alsopossess new characteristics, such as anticoagulant activity.

SUMMARY OF THE INVENTION

The present approach to studying the structural properties associatedwith the anticoagulant properties of polysaccharides was first to choosepolymers possessing well-defined chemical groups consisting of regularrepeating units, and secondly to modify their chemical structure.

Such molecules must therefore:

(1) Contain regular sequences of monomeric units, and

(2) Be chemically modifiable without destroying their structure.

Hyaluronic acid, the major component of the mammalian extracellularmatrix, consists of alternating units of N-acetylglucosamine andglucuronic acid residues, and therefore seems a suitable macromolecule.

The sulfation of alcoholic hydroxyls present in the polymeric chain of apolysaccharide or of one of its semisynthetic derivatives by the use ofa suitable sulfating agent can lead to the formation of new derivativeswith chemical-physical characteristics, but most of all biologicalcharacteristics, which are different from those of the startingmaterial.

The polyelectrolyte polyssaccharides which can be used as substrates inthe present invention include glycosaminoglycans. First and foremostamong these is hyaluronic acid and the semisynthetic derivativesthereof. Some particularly important semisynthetic derivatives ofhyaluronic acid are esters thereof with alcohols of the aliphatic,araliphatic, heterocyclic and cycloaliphatic series, designated “HYAFF,”that are described in U.S. Pat. Nos. 4,851,521, 4,965,353, and5,202,431, and EP 0 216 453. Sulfation of such pre-processedbiomaterials is a novel feature of the present invention. In this case,the sulfation reaction no longer occurs in the homogeneous phase, butrather on the surface of the biomaterial in the heterogeneous phase,activating the exposed hydroxyl groups toward the reaction solvent.

The degree of sulfation that can be obtained directly on the biomaterialis an important characteristic, and requires careful kinetic control. Toavoid the solubilization of the biomaterial, induced by the increasedhydrophilic nature of the polymer which constitutes the matrix, thenumber of —SO₃ groups per dimeric unit must not exceed a certain level,generally less than 1.5-2, depending upon the degree of hydrophilicityof the starting biomaterial. For example, in the case of HYAFF 11 films,wherein all the carboxyls are involved in ester bonding with benzylgroups, the maximum degree of sulfation should not exceed 1.5.

The reagents commonly used for sulfation include the complex betweensulfur trioxide and pyridine (SO₃-pyridine).

The reaction is conducted by adding the sulfating reagent to atetrabutylammonium salt of a polysaccharide in solution, or to asolution of a polysaccharide ester, which, in the case of partialesters, contains the remaining carboxy functions in the form oftetrabutylammonium salts, in aprotic solvents such as dimethylsulfoxide,N,N′-dimethylformamide, and N-methylpyrrolidone in the temperature rangeof from about 0° C. to about 60° C.

Different degrees of Bulfation, measured by the number of sulfate groupsper disaccharide unit, are obtained by varying the quantity ofSO₃-pyridine. The ratio between moles of hydroxyls and moles ofsulfating reagent can vary between 1:1 and 1:12.

Surprisingly, the present inventors succeeded in sulfating thepolysaccharide chain of hyiluronic acid and its semisyntheticderivatives in a specific and homogeneous manner without causing loss ofthe polymer's characteristics, in particular its molecular weight, thusobtaining new polymers with biological and physico-chemicalcharacteristics which hyaluronic acid and its semisynthetic derivativesdid not previously possess.

By this method, it is possible to obtain new polymers with differentlevels of sulfation, but with the same molecular weight. Polymers withnew biological characteristics can be obtained by using as startingmaterials biopolymers wherein the carboxy groups are salified withtetrabutylammonium salt. Such biopolymrs are not hemolytic.

A notable characteristic of these sulfated polysaccharides is theirability to increase blood coagulation time. The thrombin time test isperformed by measuring how long it takes for fibrinogen to turn tofibrin once thrombin has been added to a sample of human blood in thepresence of the test material. The thrombin time test in the same bloodsample, but in the presence of the polymer used as starting material, istaken as a reference value. The test loses significance at over 240seconds. The coagulation time is determined by simply measuring the timetaken for a sample of human blood to coagulate in the presence of thetest material. Times exceeding two hours are not considered.

Using the new biopolymers of the present invention, it is possible todevelop new biomaterials for use in the biomedical, health-care, andpharmaceutical fields. The products obtained possess biocompatible andbiological characteristics such as antithrombotic, anticoagulant, andantiviral activities. For example, sulfated polyanions have been shownto exhibit antiviral activity, including HIV inhibition. The newbiopolymers of the present invention can also be used to advantage incell growth processes, in controlled drug release systems, and moregenerally, in internal surgery, in extracorporeal oxygen circulation, inadhesion prevention, in permanent and biodegradable implants, and indialysis.

For example, as in the case of other sulfated polymers, such asdextrans, sulfated hyaluronic acid having a molecular weight in therange of between about 10,000 and about 50,000 Daltons inhibits theproduction of tumor necrosis factor (TNF), which is the main target inthe proliferation of inflammatory cells. Sulfated hyaluronic acid cantherefore be used as a local anti-inflammatory agent in the form ofhyaluroric acid-based biomaterials or compositions.

The new polymers can therefore be prepared in the form of gels, creams,or ointments, and can be used to produce biomaterials in the form ofthreads, sponges, gauzes, membranes, guide channels, non-woven fabricsand microspheres, according to the therapeutic uses for which they areintended. Lastly, depending upon the degree of sulfation and themolecular weight of the polymer, it is possible to produce polymersexhibiting antiviral activity and/or which can be use to intervene inthe various stages of cell interactions. These biopolymers can also beused in coating processes, lending new biological properties to thesurface of support material such as biomedical objects and devices.

Such sulfated biomaterials can be employed in applications where theproduct comes into contact with the blood or highly vascularizedtissues, e.g., the use of biopolymeric dialysis tubes or membranes forinternal or external surgery, which are capable of reducing celladhesion, etc. In particular, the new, soluble sulfated hyaluronic acidderivatives of the present invention can be employed in the wide varietyof applications already well known in the art for hyaluronic acid-basedbiomaterials.

For example, while hyaluronic acid derivatives having a degree ofsulfation greater than 2.5 exhibit good anticoagulant activity, themolecular weight of the starting polymer can also be significant ininfluencing the properties of the new sulfated biopolymers of thepresent invention.

In particular, at least four sulfated hyaluronic acid derivatives arenotable due to their molecular weight and degree of sulfation. Theseare:

1. Hyaluronic acid having a molecular weight in the range between about10,000 and about 50,000 Daltons, and having a degree of sulfation of2.5, 30, or 3.5;

2. Hyaluronic acid having a molecular weight in the range between about50,000 and about 250,000 Daltons, and having a degree of sulfation of2.5, 3.0, or 3.5;

3. Hyaluronic acid having a molecular weight in the range between about250,000 and about 750,000 Daltons, and having a degree of sulfation of2.5, 3.0, or 3.5; and

4. Hyaluronic acid having a molecular weight in the range between about750,000 and about l,250,000 Daltons, and having a degree of sulfation of2.5, 3.0, or 3.5.

The hyaluronic acid fractions having the molecular weights describedabove can be obtained by the use of membranes with particular molecularweight cut-off points, as is known in the art.

Among the semisynthetic ester derivatives of hyaluronic acid, polymericmatrices of HYAFF 11 (100% benzyl ester of hyaluronic acid) sulfated todegrees of 1.0 and 1.5, and HYAFF 11p75 (75% benzyl ester cf hyaluronicacid) sulfated to degrees of 0.5 and 1.0, are particularly interesting.

Further scope of the applicability of the present invention will becomeapparent from the detailed description and drawings provided below.However, it should be understood that the detailed description andspecific examples, while indicating preferred embodiments of the presentinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features, and advantages of the presentinvention will be better understood from the following detaileddescriptions taken in conjunction with the accompanying drawings, all ofwhich are given by way of illustration only, and which are notlimitative of the present invention, in which:

FIG. 1 shows the effect of hyaluronic acid sulfated with 2.0, 2.5, 3.0,and 3.5 SO₃ groups per repetitive unit on whole blood clotting time(WBCT) and thrombin time (TT).

FIG. 2 shows the growth of human umbilical vein endothelial cells incontrol medium (♦), sulfated hyaluronic acid-containing medium (▪), andhyaluronic acid-containing medium (▴) as described in Example 14.

FIG. 3 is a schematic representation of a dish prepared for thegelatin-agarose test described in Example 15. Top: a cross-sectionshowing a central well and two adjacent wells located 2 mm away. TheBACE is placed in the central well, and the test material and thecontrol are placed in the adjacent wells. Bottom: dish ready for thetest. A fourth well containing BACE is placed about 2 cm away from thethree aligned wells (proportions of distances not maintained in thefigure). The fourth well is far removed from the influence of the istest material, and is utilized as a control to assure that the migrationof BACE outside the well occurs as a uniform halo when no treatment isapplied.

FIGS. 4A, 4B, 5A, 5B, 6A, and 6B illustrate the results of theassessment of induction of angiogenesis in vitro described in Example15. FIGS. 4A and 4B show the preferential migration of endothelial cellstowards Cu(II)-sulfated hyaluronic acid rather than towards sulfatedhyaluronic acid alone. FIGs. 5A and 5B show the preferential migrationof endothelial cells towards Cu(II)-heparin rather than towards heparinalone. FIGS. 6A and 6B show that there is no preferential migration ofendothelial cells towards the Cu(II)-Tris complex rather than towardsthe medium alone.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the invention is provided to aidthose skilled in the art in practicing the present invention. Even so,the following detailed description should not be construed to undulylimit the present invention, as modifications and variations in theembodiments discussed herein may be made by those of ordinary skill inthe art without departing from the spirit or scope of the presentinventive discovery.

The contents of each of the references cited herein are hereinincorporated by reference in their entirety.

Presented below for illustrative purposes are some examples of thepreparation of new sulfated polymers according to the present invention.While these Examples are directed to hyaluronic acid and itssemisynthetic derivatives such as tetrabutylammonium salts and esters,the same methods can be applied to other polysaccharides such as otherglycosaminoglycans, alginic acid, gellan, carboxymethylcellulose,carboxy-methylamide, and carboxymethylchitin, and semisyntheticderivatives thereof, such as their tetrabutylammonium salts and partialesters with aliphatic, araliphatic, heterocyclic and cycloaliphaticalcohols, as described in U.S. Pat. Nos. 4,851,521, 5,122,598,5,300,493, 5,332,809, and 5,336,668; European Patent Application No.93917681.4; EP 0 216 453, EP 0 251 905, EP 0 342 557, EP 0 518 710, EP 0603 264, and EP 0 605 478; and WO 93/06136 and WO 94/03499.

EXAMPLE 1 Sulfation of Sodium Hyaluronate, Sulfation Degree 3

0.250 grams of the tetrabutylammonium salt of hyaluronic acid aresolubilized in 10 ml of dimethylformamide (DMF). 1.305 grams ofSO₃-pyridine solubilized in 10 ml of DMF are added to this solutionunder a flow of nitrogen. The solution is shaken for an hour at atemperature of between 4° C. and 0° C. About 200 ml of purified water,chilled to 0° C., are subsequently added. The pH of the mixture isbrought to a value of between 8.5 and 9.5 by adding 1M sodium hydroxide.The derivative is then precipitated with 120 ml of ethyl alcohol. Sodiumacetate is added to saturation, and the precipitate is left to depositfor between 1 and 24 hours at a temperature of between 0° C. and 4° C.The precipitate is separated by centrifugation, for example for 15minutes at 1,500 rpm, solubilized in purified H₂O, and then dialyzeduntil all residue reagent and reaction products have been completelyeliminated. The degree of sulfation is determined by nuclear magneticresonance (NMR).

Thrombin time and coagulation time in this and the following exampleswere determined as described in WO 92/11294. The product thus obtainedhas a thrombin time of 42.2 compared to the 11.3 seconds of the startingpolymer, and a coagulation time of over 2 hours compared to 28 minutesmeasured in the control blood.

EXAMPLE 2 Sulfation of Sodium Hyaluronate, Sulfation Degree 3.5

0.250 grams of the tetrabutylammonium salt of hyaluronic acid aresolubilized in 10 ml of dimethylformamide (DMF). 2.088 grams ofSO₃-pyridine solubilized in 10 ml of DMF are added to this solutionunder a flow of nitrogen. The solution is shaken for at least an hour ata temperature of between 4° C. and 0° C. About 200 ml of H₂O, chilled to0° C., are subsequently added. The pH of the mixture is brought to avalue of between 8.5 and 9.5 by adding 1M sodium hydroxide. Thederivative is then precipitated with 120 ml of ethyl alcohol. Anhydroussodium acetate is added to saturation, and the precipitate is left todeposit for between 1 and 24 hours at a temperature of between 4° C. and0° C. The precipitate is separated by centrifugation, for example for 15minutes at 1,500 rpm, solubilized in purified H₂O, and then dialyzeduntil all residue reagent and reaction products have been completelyeliminated. The degree of sulfation is determined by nuclear magneticresonance (NMR).

The product thus obtained has an infinite thrombin time, compared to11.3 seconds for the starting polymer.

EXAMPLE 3 Sulfation of the Partial Ethyl Ester of Hyaluronic Acid: 75%of the Carboxy Groups are in the Form of the Ethyl Ester, SulfationDegree 3

0.250 grams of the tetrabutylammonium salt of the 75% partial ethylester of hyaluronic acid (HYAFF-7p75) are solubilized in 10 ml ofdimethylformamide (DMF). 1.305 grams of SO₃-pyridine solubilized in 10ml of dimethylsulfoxide (DMSO) are added to this solution under a flowof nitrogen. The solution is shaken for at least an hour at atemperature of between 4° C. and 0° C. About 200 ml of H₂O, chilled to0° C., are subsequently added. The pH of the mixture is brought to avalue of between 8.5 and 9.5 by adding 1M sodium hydroxide. Thederivative is then precipitated with 120 ml of ethyl alcohol. Anhydroussodium acetate is added to saturation, and the precipitate is left todeposit for between 1 and 24 hours at a temperature of between 4° C. and0° C. The precipitate is separated by centrifugation, for example for 15minutes at 1,500 rpm, solubilized in purified H₂O, and then dialyzeduntil all residue reagent and reaction products have been completelyeliminated. The degree of sulfation is determined by NMR.

The product thus obtained has a thrombin time of 45 seconds, compared to11.3 seconds for the starting polymer, and a coagulation time of over 2hours compared, to 28 minutes for tho control blood.

EXAMPLE 4 Sulfation of the Partial Ethyl Ester of Hyaluronic Acid: 50%of the Carboxy Groups are in the Form of an Ethyl Ester, SulfationDegree 2.5

0.250 grams of the tetrabutylammonium salt of the 50% partial ethylester of hyaluronic acid (HYAFF-7p50, 50% of the carboxy groupsesterified with ethanol) are solubilized in 10 ml of dimethylformamide(DMF). 1.044 grams et SO₃-pyridine solubilized in 10 ml ofdimethylsulfoxide (DMSO) are added to this solution under a flow ofnitrogen. The solution is shaken for at least an hour at a temperatureof between 4° C. and 0° C. About 200 ml of H₂O, chilled to 0° C., aresubsequently added. The pH of the mixture is brought to a value ofbetween 8.5 and 9.5 by adding 1M sodium hydroxide. The derivative isthen precipitated with 120 ml of ethyl alcohol. Anhydrous sodium acetateis added to saturation and the precipitate is left to deposit forbetween 1 and 24 hours at a temperature of between 4° C. and 0° C. Theprecipitate is separated by centrifugation, for example for 15 minutesat 1,500 rpm, solubilized in purified H₂O, and then dialyzed until allresidue reagent and reaction products have been completely eliminated.The degree of sulfation is determined by NMR.

The product thus obtained has a thrombin time of 47 seconds, compared to11.3 seconds for the starting polymer, and a coagulation time of over 2hours, compared to 28 minutes for the control blood.

EXAMPLE 5 Sulfation of the Partial Ethyl Ester of Hyaluronic Acid: 25%of the Carboxy Groups are in the Form of an Ethyl Ester, SulfationDegree 2

0.250 grams of the TBA salt of a partial ethyl ester of hyaluronic acid(HYAFF-7p25, 25% of the carboxy groups esterified with ethanol) aresolubilized in 10 ml of dimethylformamide (DMF). 0.783 grams ofSO₃-pyridine solubilized in 10 ml of dimethylsulfoxide (DMSO) are addedto this solution under a flow of nitrogen. The solution is shaken for atleast an hour at a temperature of between 4° C. and 0° C. About 200 mlof H₂O, chilled to 0° C., are subsequently added. The pH of the mixtureis brought to a value of between 8.5 and 9.5 by adding 1M sodiumhydroxide. The derivative is then precipitated with 120 ml of ethylalcohol. Anhydrous sodium acetate is added to saturation, and theprecipitate is left to deposit for between 1 and 24 hours at atemperature of between 4° C. and 0° C. The precipitate is separated bycentrifugation, for example for 15 minutes at 1,500 rpm, solubilized inpurified H₂O, and then dialyzed until all residue reagent and reactionproducts have been completely eliminated. The degree of sulfation isdetermined by NMR.

The product thus obtained has a thrombin time of 49 seconds, compared to11.3 seconds for the starting polymer, and a coagulation time of over 2hours, compared to 28 minutes for the control blood.

EXAMPLE 6 Sulfation of the Partial Benzyl Ester of Hyaluronic Acid: 75%of the Carboxy Groups are in the Form of a Benzyl Ester, SulfationDegree 3.5

0.250 grams of the tetrabutylammonium salt of a partial ethyl ester ofhyaluronic acid (HYAFF-11p75, 75% of the carboxy groups esterified withbenzyl alcohol) are solubilized in 10 ml of dimethylformamide (DMF)2.088 grams of SO₃-pyridine solubilized in 10 ml of dimethylsulfoxide(DMSO) are added to this solution under a flow of nitrogen. The solutionis shaken for at least an hour at a temperature of between 4° C. and 0°C. About 200 ml of H₂O, chilled to 0° C., are subsequently added. The pHof the mixture is brought to a value of between 8.5 and 9.5 by adding 1Msodium hydroxide. The derivative is then precipitated with 120 ml ofethyl alcohol. Anhydrous sodium acetate is added to saturation, and theprecipitate is left to deposit for between 1 and 24 hours at atemperature of between 4° C. and 0° C. The precipitate is separated bycentrifugation, for example for 15 minutes at 1,500 rpm, solubilized inpurified H₂O, and then dialyzed until all residue reagent and reactionproducts have been completely eliminated. The degree of sulfacion isdetermined by NMR.

The product thus obtained has a thrombin time of 44 seconds, compared to11.3 seconds for the starting polymer, and a coagulation time of over 2hours, compared to 28 minutes for the control blood.

EXAMPLE 7 Sulfation of the Partial Benzyl Ester of Hyaluronic Acid: 50%of the Carboxy Groups are in the Form of a benzyl ester, SulfationDegree 3

0.250 grams of the tetrabutylammonium salt of a partial ethyl ester ofhyaluronic acid (HYAFF-11p50, 50% of the carboxy groups esterified withbenzyl alcohol) are solubilized in 10 ml of dimethylformamide (DMF)1.305 grams of SO₃-pyridine solubilized in 10 ml of dimethylsulfoxide(DMSO) are added to this solution under a flow of nitrogen. The solutionis shaken for at least an hour at a temperature of between 4° C. and 0°C. About 200 ml of H₂O, chilled to 0° C., are subsequently added. The pHof the mixture is brought to a value of between 8.5 and 9.5 by adding 1Msodium hydroxide. The derivative is then precipitated with 120 ml ofethyl alcohol. Anhydrous sodium acetate is added to saturation and theprecipitate is left to deposit for between 1 and 24 hours at atemperature of between 4° C. and 0° C. The precipitate is separated bycentrifugation, for example for 15 minutes at 1,500 rpm, solubilized inpurified H₂O, and then dialyzed until all residue reagent and reactionproducts have been completely eliminated. The degree of sulfation isdetermined by NMR.

The product thus obtained has a thrombin time of 46 seconds, compared to11.3 seconds for the starting polymer, and a coagulation time of over 2hours, compared to 28 minutes for the control blood.

EXAMPLE 8 Sulfation of the Partial Benzyl Ester of Hyaluronic Acid: 25%of the Carboxy Groups are in the Form of a Benzyl Ester, SulfationDegree 2

0.250 grams of the tetrabutylammonium salt of a partial ethyl ester ofhyaluronic acid (HYAFF-11p25, 25% of the carboxy groups esterified withbenzyl alcohol) are solubilized in 10 ml of dimethylformamide (DMF).0.522 grams of SO₃-pyridine solubilized in 10 ml of dimethylsulfoxide(DMSO) are added to this solution under a flow of nitrogen. The solutionis shaken for at least an hour at a temperature of between 4° C. and 0°C. About 200 ml of H₂O, chilled to 0° C., are subsequently added. The pHof the mixture is brought to a value of between 8.5 and 9.5 by adding 1Msodium hydroxide. The derivative is then precipitated with 120 ml ofethyl alcohol. Anhydrous sodium acetate is added to saturations and theprecipitate is left to deposit for between 1 and 24 hours at atemperature of between 4° C. and 0° C. The precipitate is separated bycentrifugation, for example for 15 minutes at 1,500 rpm, solubilized inpurified H₂O, and then dialyzed until all residue reagent and reactionproducts have been completely eliminated. The degree of sulfation isdetermined by NMR.

The product thus obtained has a thrombin time of 48 seconds, compared to11.3 seconds for the starting polymer, and a coagulation time of over 2hours, compared to 28 minutes for the control blood.

EXAMPLE 9 Preparation of Films of HYAFF 11, Sulfation Degree 1.5

0.250 grams of a film of HYAFF 11 are immersed in a bath of 250 ml of amixture of chloroform:dimethylformamide in a ratio of 1:1. 50 ml of asolution obtained by solubilizing 3.4 grams of a complex of pyridine-SO₃in dimethylformamide are then added.

The reaction is allowed to proceed for 2 hours at ambient temperature,after which the film is removed and then immersed in a bath of distilledwater (100 ml), and lastly in a solution of water:ethanol, 50:50. Thefilm is then oven-dried for 48 hours at 55° C.

EXAMPLE 10 Preparation of films of HYAFF 11p75, Sulfation Degree 1

0.250 grams of a film of HYAFF 11p75 are immersed in abath of 250 ml ofa mixture of chloroform:dimethylformamide in a ratio of 1:1. 50 ml of asolution obtained by solubilizing 2.3 grams of a complex of pyridine-SO₃in dimethylformamide are then added.

The reaction is allowed to proceed for 2 hours at ambient temperature,after which the film is removed and then immersed in a bath of distilledwater (about 100 ml), and lastly in a solution of water:ethanol, 50:50.The film is oven-dried for 48 hours at 55° C.

EXAMPLE 11 Biological Characterization of Soluble Sulfated HyaluronicAcid and Hyaluronic Acid Esters

Whole Blood Clotting Time in the Presence of Sulfated Hyaluronic AcidHaving Different Degrees of Sulfation

This test was performed on hyaluronic acid and sulfated hyaluronic acidusing blood from a single donor. The control contained blood alone.

For each test, three test tubes each containing 5 ml of blood wereprepared. The first constituted the blank, while in the second andthird, 25 mg of hyaluronic acid and 25 mg of sulfated hyaluronic acidwere solubilized, respectively.

The results are shown in FIG. 1, where it can be seen that hyaluronicacid having 3.0 and 3.5 SO₃ groups per repetitive unit resulted in wholeblood clotting times (WBCT) of infinity. Clotting time for whole bloodcontrols was approximately 15 minutes. Blood in the presence ofhyaluronic acid clotted after 45 minutes.

Thrombin Time in the Presence of Sulfated Hyaluronic Acid HavingDifferent Degrees of Sulfation

The thrombin time for hyaluronic acid having different degrees ofsulfation was determined using an Elvi 820 Digiclot (Logos S.p.A, Milan,Italy). This device has an incubation plate set at a temperature of 37°C., and accomodates 32 test tubes and four reagent vials, two of whichcan be magnetically stirred at 600 rpm. It contains two thermostaticmeasuring wells, fitted with a magnetic stirrer at 300 rpm, and alight-proof lid. A magnetic pipette with adaptable volumes (0.1-0.2 ml)for reagent distribution activates the device, which is stopped by eventhe slightest variations in optical density with regard to clotformation. Clotting is monitored photometrically. A ray of light from alamp first passes through a 525 nm interference filter, and lastly acapacity cell. A photodiode measures the variations in optical densityof the plasma on clot formation. A photometric signal processor stopsthe digital chronometer at the nearest tenth of a second. The throbmintime test is performed using the reagent “Trombina” (Boehringer MannheimGmbH Diagnostica).

The test is carried out on all samples using plasma obtained bycentrifugation of blood from several donors (plasma pool) which hadpreviously been treated with an anticoagulant (1 ml of a solution ofsodium citrate/9 ml of blood). Solutions were prepared at concentrationsof 1 mg/ml of hyaluronic acid and sulfated hyaluranic acid in phosphatebuffer solution.

As summarized in FIG. 1, hyaluronic acid having 2.5, 3.0, and 3.5 SO₃groups per repetitive unit lengthens the thrombin time. Hyaluronic acidhaving 2.0 SO₃ groups per repetitive unit did not lengthen the thrombintime, i.e., the thrombin time equalled that in the control, thusindicating that this particular sulfated hyaluronic acid derivative doesnot have heparin-like anticoagulant activity. Thrombin time in thepresence of hyaluronic acid is similar to that in the control.

Also shown in FIG. 1 is the quantity of heparin corresponding to 1 mg ofsulfated hyaluronic acid product, determined by means of a calibrationcurve.

Tharombin Time in the Presence of Sulfated Hyaluronic Acid Eaters HavingDifferent Decrees of Sulfation

Thrombin time was also determined on plasma in which sulfatedderivatives of hyaluronic acid is (hyaluronic acid molecularweight=200,000 Daltons) i.e., HYAFF 11 (100% benzyl ester of hyaluronicacid; sulfation degree 2.0), HYAFF 11p25 (25% benzyl ester of hyaluronicacid; sulfation degree 3.0), and HYAFF 11p75 (75% benzyl ester ofhyaluronic acid; sulfation degree 3.5) had been solubilized.

In the case of sulfated HYAFF 11, the influence of the concentrationthereof, and of thrombin, on TT was investigated.

The results for sulfated HYAFF 11 are shown in Table 1, where hyaluronicacid was used as a reference as it is soluble in plasma, and whereinthrombin concentration is in International Units (UI).

TABLE 1 THROMBIN TIME IN THE PRESENCE OF SULFATED HYAFF 11 SOLUBLEQUANTITY [] THROMBIN MATERIAL mg/ml THROMBIN TIME Plasma — ≈6 13 secSulfated HYAFF 11 8 ≈6 1 min 25 sec Hyaluronic acid 8 ≈6 30 sec SulfatedHYAFF 11 8   ≈0.6 3 min Hyaluronic acid 8   ≈0.6 50 sec Sulfated HYAFF11 2 ≈6 18 sec Hyaluronic acid 2 ≈6 17 sec

These results disclose a longer thrombin time for plasma in the presenceof sulfated HYAFF 11 than in the presence of hyaluronic acid. Theinfluence of the concentrations of hyaluronic acid, sulfated hyaluronicacid, and thrombin should be noted. Sulfated HYAFF 11 (8 mg/ml)significantly prolonged thrombin time when thrombin is employed ateither 6 UI or 0.6 UI as compared to hyaluronic acid. Low quantities (2mg/ml) of sulfated HYAFF 11 do not result in any significant variationin thrombin time.

Table 2 shows the results for sulfated HYAFF 11p25 and sulfated HYAFF11p75 on thrombin time.

TABLE 2 THROMBIN TIME IN THE PRESENCE OF SULFATED HYAFF 11p25 ANDSULFATED HYAFF 11p75 SOLUBLE MATERIAL QUANTITY THROMBIN TIME Plasma —10.3 sec HYAFF 11p75 SO₃ 5 mg/ml 12.4 sec HYAFP 11p25 SO₃ 1 mg/ml 19.4sec

The data in Table 2 demonstrate that both sulfated HYAFF 11p25 andsulfated HYAFF 11p75 prolong thrombin time. The longer thrombin time forsulfated HYAFF 11p75 corresponds to about 0.15 UI/ml of heparinactivity. The longer thrombin time for sulfated HYAFF 11p25 correspondsto about 0.25 UI/ml of heparin activity.

Reptilase Time

Reptilase is an enzyme found in the venom of Bothrox atrops that clotsfibrinogen by splitting off its fibrinopeptide A.

Reptilase time is determined by dissolving sulfated hyaluronic acid orsulfated hyaluronic acid derivative in 1 ml of 0.1 M phosphate bufferedsaline, 0.3 ml of which is then added to 0.3 ml of human plasma. Thereptilase time is determined by incubating the human plasma containingthe sulfated hyaluronic acid or derivative at 37° C. for two minutes,then adding Reptilase Reactive (fraction of thrombin extracts fromBothrox atrops venom, Hemodiagnostica Diagnostica Stago, BoehringerMannheim), and measuring the clotting time automatically (Elvi Digiclot2 Coagulometer, Logos S.p.A., Milan, Italy).

Table 3 shows the effects of the sulfated HYAFF 11, the sulfated HYAFF11p25, and the sulfated HYAFF 11p75 on reptilase time.

TABLE 3 REPTILASE TIME IN THE PRENSENCE OF SULFATED HYAFF 11, SULFATEDHYAFF 11p25, AND SULFATED HYAFF 11p75 SOLUBLE MATERIAL QUANTITYREPTILASE TIME Plasma — 15 sec Sulfated HYAFF 11 8 mg/ml 15 sec HYAFF11p75 SO₃ 5 mg/ml 15 sec HYAFF 11p25 SO₃ 1 mg/ml 15 sec

The data in Table 3 show that none of the sulfated hyaluronic acidderivatives had any significant effect on reptilase time.

EXAMPLE 12 Hemolysis Test

The hemolysis assay measures the direct interaction of substances withthe plasma membrane of erythrocytes.

25 mg of sulfated hyaluronic acid were dissolved in 0.5 ml of sodiumcitrate. The assay tube was then filled with 5 ml of fresh human blood.The control contained whole citrated blood only. The hemolysis test wascarried out as described in Albanese et al. (1994) Biomaterials 15:129.

The results obtained with sulfated hyaluronic acid show that thismaterial does not exhibit any hemolytic activity.

EXAMPLE 13 Biological Characterization of Insoluble Sulfated HyaluronicAcid Derivatives

Thrombin Time in the Presence of Insoluble Films of Sulfated HyaluronicAcid Esters Having Different Degrees of Sulfation

The thrombin time test was performed on rounds of insoluble films ofsulfated hyaluronic acid esters used to line cuvettes, essentially asdescribed in Example 11 for sulfated hyaluronic acid having differentdegrees of sulfation. 1.2 ml of plasma were added to each cuvette, whichwas then incubated together with the film rounds for 10 minutes. 0.2 mlof thrombin reagent was then added, and the clotting time was monitored.Molecular weight of hyaluronic acid and degree of sulfation of theesters were as in Example 11.

The results are shown in Table 4.

TABLE 4 THROMBIN TIMES OF HUMAN PLASMA PLACED IN CONTACT WITH FILMS OFINSOLUBLE SULFATED HYALURONIC ACID ESTERS INSOLUBLE [] THROMBIN MATERIALQUANTITY THROMBIN TIME Plasma — ≈6  9.7 sec 10.0 sec HYAFF 11p75 SO₃0.044 gr ≈6  8.3 sec  8.8 sec HYAFF 11p75 0.044 gr ≈6 11.0 sec 10.9 secHYAFF 11p75 SO₃ 0.031 gr ≈5 18.7 sec 20.9 sec HYAFF 11p75 0.031 gr ≈617.9 sec 18.1 sec HYAFF 11p75 SO₃ 0.031 gr   ≈1.5 12.3 sec 13.1 secHYAFF 11p75 0.031 gr   ≈1.5 12.6 sec 11.0 sec HYAFF 11 0.031 gr ≈6 15.6sec 17.0 sec

The data in Table 4 reveal no significant variations in the thrombintimes of plasma placed in contact with films of sulfated hyaluronic acidesters.

EXAMPLE 14 Growth of Cultured Human Umbilical Vein Endothelial Cells inthe Presence of Sulfated Hyaluroic Acid

Human umbilical vein endothelial cells were isolated from umbilicalcords by collagenase digestion following a standard protocol. The cellswere maintained in a 5% CO₂ atmosphere at 37° C. in Medium 199 (GIBCOLaboratories) with 20% fetal calf serum, L-glutamine, and gentamicin.

The endothelial cells were identified as such by their polygonalmorphology. For proliferation experiments, cells were used when cultureshad reached confluence. Hyaluronic acid was dissolved in Medium 199until a concentration of 5 mg/ml was obtained. The assay was planned inorder to allow contact periods of 24, 48, and 72 hours between thematerial and the cells. Every 24 hours the medium was removed from thewells and sterile PBS solution was rinsed over the film to remove theunattached cells. The cells were analyzed with an inverted microscope(DIAPHOT TMD Nikon) and pictures taken with a Nikon camera. The cellswere then detached with trypsin and counted in a Burker chamber. TrypanBlue was used to distinguish between dead and live cells.

FIG. 2 shows the human umbilical vein endothelial cells (HUVEC) growthcurves.

The number of endothelial cells in medium containing sulfated hyaluronicacid increased with time, and better growth is shown than in mediumcontaining hyaluronic acid or in a pure medium control.

The morphology of endothelial cells was examined using invertedmicroscopy. Endothelial cells in medium containing sulfated hyaluronicacid were well spread, with morphological alteration and withoutstructural changes in cell organization.

The same morphology was noted for the endothelial cells in the presenceof hyaluronic acid and for the control. The only remarkable differencewas in the cell proliferation. In fact, after one day, the cells in themedium containing sulfated hyaluronic acid were almost a confluentmonolayer, while the cells in medium containing hyaluronic acid or puremedium reached confluency only after three days.

EXAMPLE 15 Assessment of Induction of Angiogenesis In Vitro

Sulfated hyaluronic acid, like heparin, forms complexes with the Cu(II)ion, having a stoichiometric composition of Cu(OH)₂L (L″=“ligand”)(Barbucci et al. (1995) Gazetta Chimica Italiana, in press) . As isknown from the literature, the Cu(II)-heparin complex exhibits anangiogenic effect (Alessandri et al. (1983) Cancer Research43:1790-1797).

The ability of sulfated hyaluronic acid to induce angiogenesis in vitrousing a cell migration method (Alessandri et al. (1983) Cancer Research43:1790-1797) was therefore investigated.

The migration of endothelial cells in agar was observed, the methodbeing schematically shown in FIG. 3. The ability of a test sample toinduce angiogenesis in vitro can be determined by the number ofendothelial cells that preferentially migrate towards the test samplerather than towards the control sample.

The cell migration test to assess angiogenesis induced by the complexCu(II)-heparin, as described in Alessandri et al., was conducted in abuffer solution of 0.1 M Tris, pH 7.5. However, in the presence of Tris,the complex formed is actually Cu(II)-Tris, not Cu(II)-heparin, so thatthe angiogenic effect observed relates to the Cu(II)-Tris complex in thepresence of heparin.

The present tests were conducted using a buffer solution of 0.1 M PBS,pH 7.4. At this pH, the Cu(II) that is not in the complex precipitatesin the form of a hydroxide. Solutions of Cu(II)-biological molecule weretherefore filtered on cellulose filters having a pore size of 0.2microns in order to eliminate the copper hydroxide precipitate beforeusing solutions for testing.

Two samples of sulfated hyaluronic acid, one with 2.0 SO₃ groups, andthe other with 3.5 SO₃ groups, per repetitive unit were analyzed.Experiments were run in replicate, and samples containing the complexesCU(II)-heparin and Cu(II)-Tris were also analyzed. In each experiment,the angiogenic effect of the complex Cu(II)-biological molecule wasassessed in comparison to that of the biological molecule alone.Specifically, Cu(II)-sulfated hyaluronic acid was compared to sulfatedhyaluronic acid, and Cu(II)-hoparin was compared to heparin. In the caseof Cu(II)-Tris, the control sample contained only medium.

As shown in FIGS. 4A, 4B, 5A, 5B, 6A, and 6E, the complexCu(II)-sulfated hyaluronic acid (3.5 SO₃ groups per repetive unit)proved capable of inducing angiogenesis in vitro to an extent similar tothat of the complex Cu(II)-heparin.

As shown in FIGS. 4A and 4B, there is a preferential migration byendothelial cells towards Cu(II)-sulfated hyaluronic acid rather thantowards sulfated hyaluronic acid alone.

In the case of heparin, endothelial cells preferentially migrate towardsthe complex Cu(II)-heparin rather than towards heparin alone (FIGS. 5Aand 5B).

The effect is more pronounced with sulfated hyaluronic acid than withheparin (compare FIGS. 4A, 5A and 4B, 5B).

On the other hand, in the case of the complex Cu(II)-Tris (FIGS. 6A and6B), there is no preferential migration of the cells towards the complexrather than towards the medium alone.

The effect of the sample containing Cu(II)-sulfated hyaluronic acid (2.0SO₃ groups per repetitive unit) was comparable to that of the complexCu(II)-Tris rather than to that of the complex Cu(II)-heparin. Thisdemonstrates that the number of SO₃ groups per repetitive unitsignificantly influences obtaining heparin-like activity in inducingangiogenesis in vitro.

EXAMPLE 16 Pharmaceutical Compositions

Pharmaceutical preparations and biomaterials comprising the new sulfatedderivatives of hyaluronic acid and other sulfated polysaccharides of thepresent invention can be administered to humans, alone or in associationwith other chemical polymers, such as polyurethane, polylactic acid,carboxymethylcellulose, carboxymethylchitin, carboxymethyl starch, andcrosslinked polymers, or hyaluronic acid esters, salts, derivatives,complexes, fragments, subunits, and/or pharmacologically acceptabledrugs, as aids in the biomedical, health care, and pharmaceuticalfields.

Because of their antithrombotic and anticoagulant activities, thebiopolymers of the present invention may be advantageously used toprepare biomaterials such as guide channels, bypasses, artificial veins,or shunts to be employed in hemodialysis, cardiology, extracorporealcirculation, and more generally, in the cardiovascular system.

The angiogenic activity of Cu(II)-sulfated hyaluronic acid complexes canbe employed in stimulating capillary growth.

It has recently been demonstrated that sulfated hyaluronic acid is apotent inhibitor of Tumor Necrosis Factor-α (TNF-α) and TNF-β (Chang etal. (1994) Journal of Leukocyte Biology 55:778-784). Thus, the sulfatedhyaluronic acid and hyaluronic acid ester products of the presentinvention can also find therapeutic use as anti-inflammatory agents inthe treatment of TNF-mediated inflammation, systemic toxicity, andrelated pathologies.

Furthermore, sulfated hyaluronic acid derivatives can be employed ascoatings for the surfaces of materials using techniques such as plasmacoating to produce devices to be used in extracorporeal circulationapplications.

The sulfated hyaluronic acid derivatives of the present invention canalso be used in the form of gauzes, threads, gels, hydrogels, sponges,membranes, non-woven tissues, and microspheres, according to thetherapeutic use for which they are intended, to promote cell growthprocesses, such as keratinocyte growth, to accelerate healing inpatients affected by bedsores, wounds, burns, and skin ulcers, or asanti-adherents in surgery.

Depending upon the degree of sulfation and the molecular weight of thepolymer, the new sulfated polysaccharides of the present invention canalso be used alone or in association with other chemical polymers, suchas those listed above, or with cross-linked polymers or hyaluronic acidesters, salts, derivatives, complexes, fragments, subunits, and/orpharmacologically acceptable drugs, for example in dermatology,ophthamology, otorhinolaryngology, odontology, gynecology, urology, andas drug delivery systems in the treatment of bacterial, mycotic, orviral infections.

Examples of combination medicaments according to the present inventioninclude:

association of sulfated hyaluronic acid and a hyaluronic acid ester,such as the benzyl or ethyl ester;

association of sulfated hyaluronic acid and a crosslinked hyaluronicacid ester;

association of sulfated hyaluronic acid and a chemical polymer such asthat listed supra;

association of sulfated hyaluronic acid and Cu(II) ions;

association of sulfated hyaluronic acid and a metal ion, such as calciumor silver;

association of sulfated hyaluronic acid and a hyaluronic acid ester,with an antiinfective agent such as a basic or non-basic antibiotic,sulfamidic, antiviral (such as acyclovir), steroid antiinflammatory(such as hydrocortisone or prednisolone), non-steroid antiinflammatory(such as indomethacin), a wound healer (such as epidermal growthfactor), an antimicrobial, an antibacterial, or a disinfectant;

association of sulfated hyaluronic acid and a crosslinked hyaluronicacid, with an antiinfective agent such as a basic or non-basicantibiotic, sulfamidic, antiviral (such as acyclovir), a steroidantiinflammatory (such as hydrocortisone or prednisolone), a non-steroidantiinflammatory (such as indomethacin), a wound healer (such asepidermal growth factor), an antimicrobial, an antibacterial, or adisinfectant.

The invention being thus described, it is obvious that the same can bemodified in various ways. Such modifications are not to be considered asdivergences from the spirit and scope of the invention, and all suchmodifications that would appear obvious to one skilled in the art areintended to come within the scope of the following claims.

What is claimed is:
 1. A biomedical product comprising sulfatedhyaluronic acid or a salt thereof; wherein the number of sulfate groupsper disaccharide unit is in the range of from 0.5 to 3.5; wherein themolecular weight range of the hyaluronic acid or salt is between about50,000 and about 1,250,000 Daltons; wherein the biomedical product isselected from the group consisting of a guide channel, a bypass, anartificial vein, a shunt, a gauze, a thread, a gel, a hydrogel, a film,a membrane, a sponge, a non-woven tissue, and a microsphere; and whereinthe biomedical product is not a structure coated with the sulfatedhyaluronic acid or salt.
 2. A biomaterial for promoting cell growthprocesses which comprises sulfated hyaluronic acid or a salt thereof,wherein the number of sulfate groups per disaccharide unit is in therange of from 0.5 to 2.5, wherein the molecular weight range of thehyaluronic acid is between about 50,000 and about 1,250,000 Daltons, andwherein the biomaterial is not a structure coated with the sulfatedhyaluronic acid or salt.
 3. The biomedical product of claim 1, whereinthe molecular weight of the sulfated hyaluronic acid or salt is betweenabout 100,000 and 1,250,000 Daltons.
 4. The biomedical product of claim1, wherein the molecular weight of the sulfated hyaluronic acid or saltis between about 200,000 and 1,250,000 Daltons.
 5. The biomaterial ofclaim 2, wherein the molecular weight of the sulfated hyaluronic acid orsalt is between about 100,000 and 1,250,000 Daltons.
 6. The biomaterialof claim 2, wherein the molecular weight of the sulfated hyaluronic acidor salt is between about 200,000 and 1,250,000 Daltons.